A multi-component fluorescence composite strategy for constructing a mesh coating pattern: Design, synthesis, cell imaging, and bioactivity study of a visual antitumor fluorescent complex

2.1. Materials and instruments

All solvents and reagents are commercially available and used without further purification. The reagents used were o-carborane (98%, RG: Guaranteed reagent), isorhamnetin (98%, RG: Guaranteed reagent), and rhodamine B (98%, RG: Guaranteed reagent), which were purchased through commercial channels such as Titan Technologies. The full wavelength absorption spectra were recorded using a Shimadzu UV-2550 spectrophotometer. The fluorescence emission spectra of the Shimadzu RF-5301pcs spectrophotometer were measured under 490 nm excitation. All optical measurements were completed at room temperature.

2.2. Synthesis

Synthesis of nido-carborane and ICR: 0.2 g of o-carborane and 0.08 g of KOH were reacted in an EtOH solution (5 mL) at 80°C for 2 h and then heated under reflux and stirred for 1 h to obtain the white solid nido-carborane. 0.27 g of isorhamnetin, 0.1 g of nido-carborane, and 0.28 g of rhodamine B were reacted in EtOH solution (5 mL) at 50°C for 30 min to obtain isorhamnetin-nido-carborane-rhodamine B (ICR).

Preparations of encapsulated-complex ICR by acrylic resin (preparation of L-ICR, E-ICR, S-ICR, and R-ICR): 150 mg of ICR and Eudragit^® L 100-55 (400 mg) were dissolved and reacted in the mixed solvent of EtOH: THF =1:1 (10 mL) for 3 h. The reaction mixture was completed, distilled, and dried directly under reduced pressure to obtain a brittle red film. The film was scraped off with a spatula and crushed to yield 570 mg of L-ICR. The same strategy was used to obtain 420 mg of E-ICR, 421 mg of S-ICR, and 437 mg of R-ICR, respectively.

2.3. UV-Vis and fluorescence spectroscopy with fluorescence stability assay

The ultraviolet-visible (UV-vis) spectra were captured using a Shimadzu UV-2550 spectrophotometer, and the fluorescence emission spectra were recorded with a Shimadzu RF-5301PCS spectrofluorometer at 25°C. To improve the solubility of the compounds in diverse solvents, the four complexes were initially dissolved in dimethyl sulfoxide (DMSO) at a concentration of 2 mg/mL. Subsequently, serial dilutions were performed in dichloromethane (DCM), MeOH, EtOH, and buffer solutions with varying pH values (pH = 4.5, 5.0, 6.5, 7.4, and 8.0) to prepare concentration gradients of 0.02 mg/mL, 0.04 mg/mL, and 0.06 mg/mL for the acquisition of UV-vis and fluorescence spectra. The UV-vis spectral measurements were conducted within the wavelength range of 400-700 nm. The excitation wavelength for fluorescence measurements was determined based on the maximum absorption wavelength in the UV region, and the corresponding fluorescence emission spectra were obtained. Substances were dissolved in Dimethyl sulfoxide (DMSO), and the stability of fluorescence intensity of the four complexes was evaluated for 5 and 10 min in buffers with varying pH values (pH = 4.5, 5.0, 6.5).

2.4. Encapsulation efficiency (EN) and drug loading (DL)

A regression standard curve of the concentration and absorbance at 550 nm for L-ICR, E-ICR, S-ICR, and R-ICR DMSO solutions was plotted to accurately determine the encapsulation efficiency and drug loading capacity of the test complexes. Appropriate amounts of the test samples were accurately weighed, and their absorbance was measured using a Shimadzu UV-2550 spectrophotometer under experimental conditions identical to those for constructing the standard curve. The encapsulation efficiency (Eq. 1) and drug loading (Eq. 2) were calculated by means of the correspondence between absorbance and drug content reflected in the standard curve.

The calculation formulas (Eqs. 1-2) are as follows:

2.5. Cell proliferation toxicity test (CCK-8)

Trypsin was used to digest the PC3 and HeLa cells in the logarithmic growth stage, resulting in a cell suspension. The concentration of the cell suspension was then adjusted to 5000 cells/well. Three multiple wells in each group of 96-well plates were inoculated with the cell suspension. The cells were cultured for 48 h, during which time the cell coverage rate per hole increased to over 80%. A blank control was established concurrently with experimental groups. Experimental groups were subsequently treated with serial concentrations (4, 8, 12, 16, 20, and 24 μg/mL) of L-ICR and E-ICR, followed by a 24-h co-incubation period. Post-treatment, 10 μL of Cell Counting Kit-8 (CCK-8) solution was dispensed into each well (with meticulous avoidance of bubble formation to prevent optical interference). Following the 1 h incubation at 37°C under 5% CO₂, absorbance was determined at 450 nm using a microplate reader. Data were recorded for subsequent analysis.

Formula (Eqs. 3-4) calculation:

2.6. Fourier transform infrared spectroscopy

Infrared spectra were acquired by the KBr pellet method. Background controls were prepared by pressing dried, ground KBr into transparent pellets under infrared illumination. Sample spectra were obtained by homogenizing dried L-CR with KBr, pelletizing, and scanning via Fourier transform infrared (FTIR) spectroscopy.

2.7. Transmission electron microscope (TEM)

Zeiss Ultra Plus TEM with an accelerating voltage of 15 keV was employed to obtain TEM images at scales of 1.0 μm, 200 nm, and 100 nm, respectively, which was equipped with an Oxford Instruments X-Max 60 mm² SDD X-ray microanalysis system.

2.8. Cell imaging

HeLa cells in the logarithmic growth phase were treated with trypsin prior to seeding them in a 96-well plate with a circular cover, incubating them in an incubator with 5% CO₂, and culturing them for 24 h at 37°C to encourage adhesion. The prepared polymer L-ICR stock solutions (10 mg/mL) were made in DMSO, respectively, and then diluted with DMSO to prepare the appropriate quantities of solution. For a full day, the cells in each sample were removed from their original culture medium and placed in a medium containing 10 μg/mL. After that, it was cleaned twice, fixed for twenty-five minutes with paraformaldehyde. The solution was incubated with anti-fluorescence quenching scaffolds, followed by staining with 4’,6-diamidino-2-phenylindole (DAPI), a nucleic acid stain, in a dark room for 25 min. After removing the repair solution, the sample was washed twice with PBS, and cell fluorescence images were acquired using a fluorescence microscope.

2.9. In vitro release test

To investigate the effect of pH on drug release rate, buffer solutions with pH 4.5, 5.5, and 6.5 were prepared using distilled water as the solvent, with disodium hydrogen phosphate (Na₂HPO₄·12H₂O) and citric acid (C₆H₈O₇·H₂O) in different ratios to simulate normal physiological and tumor microenvironment pH conditions. L-ICR ethanol solutions at concentrations of 3.33, 6.67, 10.00, 13.30, and 16.70 μg/mL were prepared. A UV spectrophotometer was used to scan each solution across the 200-800 nm wavelength range, measuring absorbance at the maximum absorption wavelength. A linear-fitted absorbance-concentration standard curve (R² > 0.998) was established for quantitative data analysis. A 2 mL aliquot of 1 mg/mL L-ICR solution was loaded into a dialysis bag (MD34-1000000), which was immersed in 40 mL of the respective pH buffers (4.5, 5.5, 6.5). In vitro release tests were conducted at 37°C with 100 rpm shaking: aliquots were sampled at 0.5, 1, 1.5, 2, 3, 4, 5, 6, 8, 10, 12, 16, 20, and 24 h, with 3 mL fresh buffer replenished post-sampling. The absorbance of L-ICR in the sampling solution was measured using a UV-Vis spectrophotometer, and the cumulative release of L-ICR was estimated based on the regression standard curve and formula. The formula (Eq. 5) is as follows:

Q: Percentage of cumulative drug release, %;

C_n: Concentration of sampled for the nth time, μɡ/mL;

V: Total volume of the release medium, 40 mL;

V_i: Sampling volume at the i th time point, 3 mL;

C_i: Sampling concentration at the i th point, μɡ/mL;

m_drug: Quality of polymer in drug carrier, μɡ.

Design

Regarding synthesis design, the scheme was primarily prepared using the continuous method. To address the solubility issue, potassium hydroxide, a strong alkali, was first applied to nido-carborane and isorhamnetin, resulting in the formation of zwitterion small molecules [22]. Next, a mixture of nido-carborane, isorhamnetin potassium salt, and rhodamine B was added to cause a reaction that resulted in a complete bond in the form of ions. In the end, it interacted with various acrylic resin kinds to produce a polymer aggregation form and a good coating effect (Scheme 1).

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Scheme 1.

Preparation routes of four fluorescent complexes.

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The results of the encapsulation efficiency and drug loading capacity calculated according to the formulas have been shown in the Table 1.

In buffers with pH 4.5 - 8.0, their maximum absorption wavelengths showed no significant shifts, demonstrating pH-insensitive absorption properties, though absorbance increased with concentration (Table 2). Emission spectra under 550 nm excitation (Table 3) revealed emission wavelengths concentrated at 566 - 77 nm, with L-ICR, S-ICR, and R-ICR displaying the highest fluorescence intensity in low-polarity solvents (DCM) and the lowest in high-polarity solvents (MeOH) due to enhanced non-radiative transitions, while pH variations had negligible effects on emission characteristics. Furthermore, fluorescence intensity of all four complexes increased with polymer concentration under identical solvent and pH conditions. At a fluorescence emission wavelength of λem = 568 nm, the fluorescence intensities of L-ICR at concentrations of 0.13, 0.27, and 0.40 μg/mL were 198.5, 332.5, and 518.8 a.u., respectively.

It can be seen from the figure (Figure 2) that the infrared spectra of L-ICR and E-ICR have a very strong absorption peak near 1600 cm (⁻¹). Based on relevant reference, it is considered that this peak belongs to the aromatic stretching vibration in the flavone backbone of isorhamnetin [23]. The subsequent absorption peak near 2500 cm⁻¹ is the stretching vibration of B-H in nido-carborane [19,24]. These findings indicate that these compounds have a good coating.

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Figure 2.

FTIR spectrum of L-ICR and E-ICR.

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To ascertain the stability of the target complex in buffers with varying pH (pH=4.5, 5.0, and 6.5), the maximum UV absorption wavelength of the complex in the corresponding buffer was chosen as the excitation wavelength (Figure 3). Both L-ICR and E-ICR exhibited continuous fluorescence stability under different pH conditions within 5 (Figure 3a-c) and 10 min (Figure 3d-f), respectively.

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Figure 3.

Fluorescence stability of L-ICR and E-ICR compounds in buffers with different pH (pH=4.5, 5.0, 6.5) at (a-c) 5 min and (d-f) 10 min.

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The CCK-8 assay results (Figure 4 and Table 4) showed that the proliferation rates of PC3 cancer cells treated with L-ICR and E-ICR were 48.25% and 64.65% (p < 0.05), respectively, while the proliferation rates of HeLa cancer cells treated with L-ICR and E-ICR were 67.66% and 74.19%, respectively. These results suggest that the anti-tumor activity of L-ICR was significantly higher than that of E-ICR and that the tumor inhibitory effect of both complex increased with the concentration of the complex. Thus, L-ICR was chosen for experiments involving cell imaging, TEM.

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Figure 4.

The proliferation rate of (a) Prostate cancer cell line 3 (PC3) and (b) Human epithelial cervical adenocarcinoma cells (HeLa) treated by L-ICR and E-ICR at 7 concentrations (0, 4, 8, 12, 16, 20, 24 µg/mL). In the figure, orange represents L-ICR and green represents E-ICR.

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TEM was used to characterize the morphological features of L-ICR. Most of the fluorescent complexes are tightly packed together and entirely covered in acrylic resin, as seen in the image (Figure 5a-f). The complex exhibits a reticular porous morphology, where the network structure enhances its surface area-to-volume ratio, thereby improving cellular uptake efficiency [25]. In addition, the acrylic resin coating serves as a diffusion barrier to slow down the drug release rate. These structural features are directly related to the observed sustained-release kinetics and prolonged in vivo efficacy, providing a mechanism basis for the enhanced therapeutic performance of this complex.

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Figure 5.

TEM images of L-ICR at different sizes: (a-f) show structural details at 1.0 μm, 500 nm, 200 nm, 200 nm,100 nm, and 100 nm scales, respectively.

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To visually observe the distribution of L-ICR in cancer cells, we performed fluorescence imaging studies on various L-ICR channels in HeLa cells (Figure 6). After being exposed to L-ICR action for 48 h, HeLa cells were stained under a fluorescence microscope and captured on camera using a confocal laser. This image provides a bright-field image, DAPI-dyed blue nuclear image, green-red channel composite image, superposition of the above four images J, and fluorescence colocalization scatterplot of DAPI versus green and red channels. Bright and significant fluorescence was produced by the complex in both channels, which served as the foundation for real-time visualization of the complex in vivo. It was evident from the combined images that the complex had good cell entry. Through fluorescence colocalization analysis (Figure 7), the Pearson’s correlation coefficients of the green channel with DAPI at pH 5.0, 5.5, and 6.5 were determined to be 0.66, 0.59, and 0.58, respectively (Figure 7a-c). For the red channel, the corresponding correlation coefficients with DAPI at these pH values were 0.37, 0.29, and 0.54 (Figure 7d-f). The Pearson’s correlation coefficients (0.66, 0.59, 0.58, 0.37, 0.29, 0.54) generated by ImageJ are all greater than 0, and most of them (0.66, 0.59, 0.58, 0.54) are greater than 0.5, indicating that L-ICR can effectively cross the biological barrier and enter the cell. The Pearson coefficient of the red channel was the lowest at pH 5.5 (0.29), indicating that the fluorophore separated and the complex was released in the environment of the endosome (pH 5.0-5.5), which is a key subcellular niche for drug endocytosis and escape [26], while the green channel maintained nuclear targeting (0.58) to ensure the efficacy of the drug.

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Figure 6.

Fluorescent microscope imaging of HeLa cells treated with L-ICR fluorescent complexes in different channels. Excitation wavelength: DAPI 340-390 nm, green channel 450-480 nm, red channel 550-590 nm.

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Figure 7.

Fluorescence colocalization scatterplot of DAPI versus green and red channels: (a) Green channel (pH = 5.0); (b) Green channel (pH = 5.5); (c) Green channel (pH = 6.5); (d) Red channel (pH = 5.0); (e) Red channel (pH = 5.5); (f) Red channel (pH = 6.5). Pearson’s R values are displayed for each condition.

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The concentration of the L-ICR solution has a linear relationship with UV absorbance, and the linear equation is a=0.0209C+0.002(R²=0.9999) (Figure 8a). The release of L-ICR under pH-varying buffers (pH=4.5, 5.5, and 6.5) (Figure 8b) demonstrated that the release rate varied depending on the circumstances. The pH=6.5 buffer solution has the fastest release rate; L-ICR is released quickly in the first 2 hours, gradually slows down in the next 2-4 hours, and then tends to stabilize after 4 hours; 85% of the total cumulative release amount is reached. The release rate of L-ICR in the buffer with pH=5.5 is the next to be discussed. It starts out quickly and slows down over the course of 0-4 h, stabilizing at 8 h. In the pH=5.5 buffer, the final cumulative release of L-ICR reaches 92%. The buffer with pH=4.5 has the slowest release; it releases slowly over the course of 0-2 h and becomes calm after that time. The final cumulative release L-ICR in the buffer reaches 48 percent. The selection of the above pH values is based on physiological environment simulation: pH 4.5 mimics the acidic gastric conditions, pH 5.5 replicates the duodenal jejunal junction (pH 5.0-6.0) [27], and pH 6.5 matches the tumor microenvironment (pH 6.0-6.8) [28,29]. Eudragit L100-55 acrylic resin, a widely used enteric coating material for tablets and capsules, exhibits a pH-dependent dissolution threshold (pH > 5.5). The experimental results indicate that this design can achieve targeted drug delivery by triggering the intestinal environment and achieving rapid drug release through the tumor microenvironment, while maintaining stability in the stomach. This mechanism provides a robust basis for the precise delivery of therapeutic agents to target lesions. More research is still needed to determine whether the compound can reach the pH of various environments in vivo and still have the targeted action and controlled release effects.

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Figure 8.

(a) Concentration absorbance standard curves and (b) cumulative drug release curvesof L-ICR in buffers with different pH (pH =4.5, 5.5, 6.5).

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